Abiotic-Stress Application, Global Berry Proteome Profiling, and Cold-Stress Tolerance in Transformed Winegrape (Vitis vinifera L.).

Wine grape (Vitis vinifera L.) was domesticated more than 7,000 years ago and continues to provide one of the most important fruit crops worldwide. V. vinifera cultivars are moderately sensitive to salt and temperature stresses, whereas moderate water deficit stress results in minimal loss in berry yields, increased berry quality, and higher rates of winter survival of vines. The molecular mechanisms controlling both the desirable and undesirable effects of these stresses are poorly understood. To lay the foundation for our current understanding into the molecular mechanisms that underlie these environmental stress responses, a survey of the state-of-the-art literature was performed (Chapter I). For elicitation of a pronounced change in abundance of transcripts in response to abiotic-stresses a recirculating drip hydroponic growth system was built using expanded clay ball media to eliminate the buffering effect of soil. This system effectively ameliorated stress-related symptoms of hypoxia exhibited by vines grown using a previously described media-free hydroponic method (Chapter II). Our understanding of the complex nature of the V. vinifera berry proteome is extremely limited. Using a phenol-based protein extraction protocol, large two-dimensional polyacrylamide gels, and separation of proteins across two overlapping isoelectic focusing (IEF) ranges spanning pI 4-11, a total of 802 proteins were identified from whole grape berries across four distinct phases (phases I-IV) of berry development including immature green berries, lag phase, veráison, and mature berries spanning modified Eichhorn and Lorennz (E-L) system stages 29-38. Abundance profiles were generated by two-dimensional hierarchical clustering for 652 proteins whose abundance profiles could be confirmed across all four phases of berry development resulting in 21 discrete clusters. Profiles were also compared between berries collected from well watered and water deficit treated vines. Fifty one proteins showed significant differences in relative abundance in response to water deficit stress at one or more developmental phases. Comparison of the current results with previously published studies indicated that 606 of the proteins identified were previously undescribed making the current study the most comprehensive ever undertaken to date. Novel insights included the discovery that several pathogenesis-related (PR) proteins appear during early berry development, seed-storage proteins reach maximal abundance during phase III, and programmed cell death (PCD) proteins and associated salvage enzymes peak during phase IV. Water deficit stress resulted in the decreased abundance of proteins associated with mitochondrial energy production, but in the increased abundance of proteins associated with pathogenesis and heat shock responses as well as photosynthetic-, ABA-induced osmotic- and nutrient deficit-stress responses and phenylpropanoid biosynthetic enzymes (Chapter III). To investigate the effect of constitutive expression of a Vitis vinifera CBF transcription factor (VvCBF4), a variety of stresses were applied to 35S:VvCBF4 transformed Vitis cv. Freedom. The VvCBF4-transformed grapes showed a dwarf phenotype and increased tolerance to freezing stress and decreased ion-leakage from leaves in a dose-response manner that correlated well with transgene expression. Investigation of changes in mRNA transcript and protein abundance patterns of one transformed line expressing VvCBF4 under unstressed control conditions revealed numerous changes, some of which could be related to the observed dwarf and cold-tolerant phenotypes (Chapter IV). Implications for future research into the potential role of VvCBF4 in abiotic stress resistance for stresses other than cold are yet to be determined, but are necessary for a full understanding of the functional role of this transcription factor. Other molecular mechanisms of abiotic stress response in V. vinifera might be revealed through further 2DGE or Difference In-Gel Electrophoresis (DIGE) analysis of Vitis tissues in conjunction with newly developed gel-free proteomics methods utilizing highly accurate and sensitive Mass Spectrometry (MS) technologies (Chapter V)